Oral cancer stem cells drive tumourigenesis through activation of stromal fibroblasts.

BACKGROUND: Cancer stem cells are responsible for tumour progression and chemoresistance. Fibroblasts surrounding a tumour also promote progression and fibroblast "activation" is an independent prognostic marker in oral cancer. Cancer stem cells may therefore promote tumourigenesis through communication with stromal fibroblasts. METHODS: Cancer stem cells were isolated from oral cancer cell lines by adherence to fibronectin or cisplatin resistance. Fibroblasts were exposed to conditioned medium from these cells, and the activation markers, alpha smooth muscle actin and interleukin-6, were assessed using qPCR and immunofluorescence. Stem cell markers and smooth muscle actin were examined in oral cancer tissue using immunohistochemistry. RESULTS: Adherent and chemoresistant cells expressed increased levels of stem cell markers CD24, CD44 and CD29 compared with unsorted cells. Adherent cells exhibited lower growth rate, higher colony forming efficiency and increased cisplatin resistance than unsorted cells. Smooth muscle actin and Interleukin-6 expression were increased in fibroblasts exposed to conditioned medium. In oral cancer tissue, there was a positive correlation between expression of αSMA and stem cell markers. CONCLUSIONS: Adherence to fibronectin and chemoresistance isolates stem-like cells that can activate fibroblasts, which together with a correlation between markers of both in vivo, provides a mechanism by which such cells drive tumourigenesis.

The chemokine lymphotactin and its recombinant variants in oral cancer cell regulation.

BACKGROUND: The expression of XCR1 receptor and its metamorphic ligand lymphotactin (hLtn) has been shown in cancers but their precise role in tumorigenesis is poorly understood including the significance of the physiologically existing hLtn monomeric (CC3) and dimeric (W55D) confirmations where the latter thought to function as the receptor antagonist. The aim of this study was to explore the functional role of bioengineered hLtn variants and the role of fibroblasts in XCR1/hLtn expression regulation in oral cancer cells (OCCL). MATERIAL AND METHODS: qRT-PCR and flow cytometry were performed to evaluate mRNA and protein expression of XCR1 and hLtn. Recombinant hLtn variants (wild-type, CC3 and W55D mutant) were designed, expressed, purified and evaluated using proliferation, adhesion and chemotaxis assays. XCR1 and hLtn expression regulation by fibroblasts was determined using indirect co-culture. XCR1 and hLtn expression in primary and metastatic OSCC tissue was assessed using immunohistochemistry. RESULTS: hLtn caused a significant decrease in OCCL XCR1 surface protein expression. hLtn CC3 mutant was highly functional facilitating proliferation and migration. Conditioned media from primary cancer-associated and senescent fibroblasts significantly upregulated XCR1 and hLtn mRNA expression in OCCL. Immunohistochemistry revealed higher XCR1 and hLtn expression in metastatic tumour deposits and surrounding stroma compared to primary OSCC tissue. CONCLUSIONS: The development of hLtn biological mutants, regulation of XCR1 expression by its ligand hLtn and crosstalk with fibroblasts are novel findings suggesting an important role for the XCR1/hLtn axis within the OSCC tumour microenvironment. These discoveries build upon previous studies and suggest that the hLtn/XCR1 axis has a significant role in stromal crosstalk and OSCC progression.

Identification of key determinants in Porphyromonas gingivalis host-cell invasion assays.

The periodontal pathogen Porphyromonas gingivalis can invade host cells, a virulence trait which may contribute to the persistence of infection at subgingival sites. Whilst the antibiotic protection assay has been commonly employed to investigate and quantify P. gingivalis invasion, data obtained have varied widely and a thorough investigation of the factors influencing this is lacking. We investigated the role of a number of bacterial and host-cell factors and report that the growth phase of P. gingivalis, source (laboratory strain vs. clinical strain), host-cell identity (cell line vs. primary), host-cell lysis method, and host-cell passage number had no significant effect on bacterial invasion. However, incubation time, host-cell seeding density, method of quantification (viable count vs. DNA), and whether host cells were plated or in suspension, were shown to influence invasion. Also, cells isolated by rapid adhesion to fibronectin exhibited higher levels of P. gingivalis invasion, possibly as a result of increased levels of active α5ß1 integrin. Interestingly, this may represent a population of cells with stem cell-like properties. This study provides important new information by identifying the most important factors that influence P. gingivalis invasion assays and may help to explain variations in the levels previously reported.

Physiological Fluid Flow Moderates Fibroblast Responses to TGF-ß1.

Fibroblasts are the major cellular component of connective tissue and experience mechanical perturbations due to matrix remodelling and interstitial fluid movement. Transforming growth factor ß1 (TGF-ß1) can promote differentiation of fibroblasts in vitro to a contractile myofibroblastic phenotype characterised by the presence of α-smooth muscle actin (α-SMA) rich stress fibres. To study the role of mechanical stimulation in this process, we examined the response of primary human fibroblasts to physiological levels of fluid movement and its influence on fibroblast differentiation and responses to TGF-ß1. We reported that in both oral and dermal fibroblasts, physiological levels of fluid flow induced widespread changes in gene expression compared to static cultures, including up-regulation of genes associated with TGFß signalling and endocytosis. TGF-ß1, activin A and markers of myofibroblast differentiation including α-SMA and collagen IA1 were also increased by flow but surprisingly the combination of flow and exogenous TGF-ß1 resulted in reduced differentiation. Our findings suggest this may result from enhanced internalisation of caveolin and TGF-ß receptor II. These findings suggest that a) low levels of fluid flow induce myofibroblast differentiation and b) fluid flow antagonises the fibroblast response to pro-differentiation signals such as TGF-ß1. We propose that this may be a novel mechanism by which mechanical forces buffer responses to chemical signals in vivo, maintaining a context-specific fibroblast phenotype. J. Cell. Biochem. 118: 878-890, 2017. © 2016 Wiley Periodicals, Inc.

The Periodontal Pathogen Porphyromonas gingivalis Preferentially Interacts with Oral Epithelial Cells in S Phase of the Cell Cycle.

Porphyromonas gingivalis, a key periodontal pathogen, is capable of invading a variety of cells, including oral keratinocytes, by exploiting host cell receptors, including alpha-5 beta-1 (α5ß1) integrin. Previous studies have shown that P. gingivalis accelerates the cell cycle and prevents apoptosis of host cells, but it is not known whether the cell cycle phases influence bacterium-cell interactions. The cell cycle distribution of oral keratinocytes was characterized by flow cytometry and BrdU (5-bromo-2-deoxyuridine) staining following synchronization of cultures by serum starvation. The effect of cell cycle phases on P. gingivalis invasion was measured by using antibiotic protection assays and flow cytometry, and these results were correlated with gene and surface expression levels of α5 integrin and urokinase plasminogen activator receptor (uPAR). There was a positive correlation (R = 0.98) between the number of cells in S phase and P. gingivalis invasion, the organism was more highly associated with cells in S phase than with cells in G2 and G1 phases, and S-phase cells contained 10 times more bacteria than did cells that were not in S phase. Our findings also show that α5 integrin, but not uPAR, was positively correlated with cells in S phase, which is consistent with previous reports indicating that P. gingivalis invasion of cells is mediated by α5 integrin. This study shows for the first time that P. gingivalis preferentially associates with and invades cells in the S phase of the cell cycle. The mechanism of targeting stable dividing cells may have implications for the treatment of periodontal diseases and may partly explain the persistence of this organism at subgingival sites.

The chemokine receptors CXCR1 and CXCR2 regulate oral cancer cell behaviour.

BACKGROUND: Chemokines regulate physiological and pathological leucocyte trafficking, and chemokine receptors play a role in tumorigenesis. Expression of interleukin-8 (IL-8) receptors CXCR1 and CXCR2 has been shown in oral squamous cell carcinoma (OSCC) but remains poorly characterised. This aim of this study was to investigate CXCR1 and CXCR2 expression on normal oral keratinocytes (NOKs) and oral cancer cell lines (OCCL) and their relative response when exposed to IL-8 and growth-related oncogene-α (which selectively binds CXCR2). METHODS: mRNA and protein expression was studied using RT-PCR, immunocytochemistry and flow cytometry. ELISAs were used to investigate ERK1/2 phosphorylation and MMP production, whereas a MTS-based assay was employed to study proliferation. Migration assays were carried out using modified Boyden chambers with a matrigel coating used for invasion assays. RESULTS: mRNA expression of CXCR1 and CXCR2 was seen in both NOKs and OCCL with significantly higher protein expression in OCCL. Exposure to IL-8 and GROα increased intracellular ERK phosphorylation, proliferation, migration and invasion with OCCL showing a greater response than NOKs. These effects were mediated through CXCR1 and CXCR2 (for IL-8) and CXCR2 (for GROα) as receptor-blocking antibodies significantly inhibited the responses. IL-8 and GROα also increased MMP-9 release from NOKs and OCCL with significantly higher amounts released by OCCL. However, an increase in MMP-7 production was only seen in OCCL. CONCLUSIONS: Functional CXCR1 and CXCR2 exist on normal and cancerous oral epithelial cells, and our data suggests a role for these receptors in oral cancer biology.

Temporal mismatch between pain behaviour, skin Nerve Growth factor and intra-epidermal nerve fibre density in trigeminal neuropathic pain.

BACKGROUND: The neurotrophin Nerve Growth factor (NGF) is known to influence the phenotype of mature nociceptors, for example by altering synthesis of neuropeptides, and changes in NGF levels have been implicated in the pathophysiology of chronic pain conditions such as neuropathic pain. We have tested the hypothesis that after partial nerve injury, NGF accumulates within the skin and causes 'pro-nociceptive' phenotypic changes in the remaining population of sensory nerve fibres, which could underpin the development of neuropathic pain. RESULTS: Eleven days after chronic constriction injury of the rat mental nerve the intra-epidermal nerve fibre density of the chin skin from had reduced from 11.6 ± 4.9 fibres/mm to 1.0 ± 0.4 fibres/mm; this slowly recovered to 2.4 ± 2.0 fibres/mm on day 14 and 4.0 ± 0.8 fibres/mm on day 21. Cold hyperalgesia in the ipsilateral lower lip was detectable 11 days after chronic constriction injury, although at this time skin [NGF] did not differ between sides. At 14 days post-injury, there was a significantly greater [NGF] ipsilaterally compared to contralaterally (ipsilateral = 111 ± 23 pg/mg, contralateral = 69 ± 13 pg/mg), but there was no behavioural evidence of neuropathic pain at this time-point. By 21 days post-injury, skin [NGF] was elevated bilaterally and there was a significant increase in the proportion of TrkA-positive (the high-affinity NGF receptor) intra-epidermal nerve fibres that were immunolabelled for the neuropeptide Calcitonin Gene-related peptide. CONCLUSIONS: The temporal mismatch in behaviour, skin [NGF] and phenotypic changes in sensory nerve fibres indicate that increased [NGF] does not cause hyperalgesia after partial mental nerve injury, although it may contribute to the altered neurochemistry of cutaneous nerve fibres.

ADAM 10 is over expressed in oral squamous cell carcinoma and contributes to invasive behaviour through a functional association with αvß6 integrin.

A disintegrin and metalloprotease (ADAM) proteins are upregulated in cancer and can interact with integrin receptors. We investigated whether such interactions may have functional significance in oral squamous cell carcinoma (OSCC). ADAM 10 expression was increased in OSCC tissue and cell lines compared to normal oral mucosa. Silencing of ADAM 10 reduced migration and invasion specifically in OSCC cells over-expressing αvß6 integrin. This may result from ADAM 10-induced up-regulation of MMPs. We conclude ADAM 10 may influence OSCC invasion by functionally interacting with αvß6 integrin which we have previously shown is over expressed in OSCC.

Differential adhesion and invasion by Staphylococcus aureus of epithelial cells derived from different anatomical sites.

Staphylococcus aureus can invade epithelial cells, and the host-cell receptor α(5)ß(1) integrin is thought to mediate this process. The aim of this study was to investigate S. aureus invasion of epithelial cell lines derived from oral (H357), skin (UP) and nasopharyngeal (Detroit 562) sites and to determine whether any differences were due to the levels of α(5)ß(1) integrin expressed. While the adhesion and invasion of two S. aureus strains were similar in both oral and skin-derived keratinocytes, this was markedly reduced in the nasopharyngeal cell line, despite it expressing similar levels of α(5)ß(1). While this might be explainable on the basis of availability of cell receptor, adhesion to and invasion of H357 and UP cells by S. aureus were enhanced when the epithelial cells were in suspension rather than on a surface, and levels of α(5) integrin subunit mRNA were also increased. Detroit 562 cells exhibited a similar α(5) gene upregulation, but this did not result in enhanced adhesion and invasion of S. aureus. The Detroit 562 cells also showed reduced adhesion to fibronectin compared with the other cell types. This, and the low S. aureus invasion, may result from reduced α(5)ß(1) integrin activity or from variation in an as-yet-unidentified additional receptor or accessory molecule. These studies shed further light on the mechanisms of S. aureus invasion of human cells.

Endothelin-1 stimulates motility of head and neck squamous carcinoma cells by promoting stromal-epithelial interactions.

The invasion and migration of cancer cells is increasingly recognised to be influenced by factors derived from adjacent tumour-associated stroma. The contextual signals regulating stromal-tumour interactions, however, remain poorly understood. Here, we identify a role for endothelin-1 (ET-1), a mitogenic peptide elevated in a number of malignancies, in promoting pro-metastatic cross-talk between head and neck cancer cells and adjacent fibroblasts. We demonstrate that treatment of oral fibroblasts with ET-1 activates ADAM17-mediated release of epidermal growth factor receptor (EGFR) ligands, triggering EGFR signalling and increased motility in neighbouring head and neck cancer cells. ET-1-mediated paracrine transactivation of EGFR also increased cyclo-oxygenase-2 levels in the cancer cells, providing a molecular insight into the mechanisms by which the elevated levels of ET-1 observed in head and neck cancers may contribute to disease progression.

The role of α9ß1 integrin in modulating epithelial cell behaviour.

BACKGROUND: Integrins initiate signalling in response to the extracellular matrix (ECM), which is important in wound healing and cancer. Previous studies have shown that over-expression of the αvß6 integrin in oral squamous cell carcinoma (OSCC) cells results in enhanced motility and expression of matrix-degrading proteases, and the aim of this study was to investigate whether this is also the case for the α9ß1 integrin. METHODS: H357 OSCCcells were transfected with the α9 integrin subunit and proliferation, adhesion and migration assays were performed on these along with null vector control and wild-type cells. The effect of ligand engagement on matrix metalloproteinase expression and the plasminogen activator system was measured using ELISA and chromogenic assays. Expression of α9 integrin was examined in oral squamous cell carcinoma tissue by immunohistochemistry. RESULTS: Functionally active α9 integrin mediated specific upregulation of adhesion and migration towards the TNfn3RAA fragment of tenascin-C but reduced proliferation. Migration towards collagen I was also enhanced in transfected cells. Matrix metalloproteinase-2 and metalloproteinase-9 expression was increased upon TNfn3RAA ligand engagement. Cell surface plasmin generation was also enhanced in α9-expressing cells and was the result of enhanced expression of urokinase receptor. In normal oral mucosa, α9 integrin expression was restricted to the suprabasal and prickle cell layers, and expression was heterogeneous in tumours but present in islands infiltrating connective tissue particularly in moderately and well-differentiated lesions. CONCLUSIONS: The α9ß1 integrin may play a key role in modulation of tumour behaviour including enhanced cell migration and expression of matrix-degrading proteases.

The αE(CD103)ß7 integrin interacts with oral and skin keratinocytes in an E-cadherin-independent manner*.

The integrin αE(CD103)ß7 (αEß7) is expressed by intraepithelial lymphocytes, dendritic cells and regulatory T cells. It plays an important role in the mucosal immune system by retaining lymphocytes within the epithelium and is involved in graft rejection, immunity against tumours and the generation of gut-homing effector cells. In gut and breast, the ligand for αEß7 is E-cadherin but in human oral mucosa and skin, there is evidence that lymphocytes use an alternative, unknown, ligand. In the present study, the I domain of the human αE subunit, which contains the E-cadherin-binding site, was locked in a highly active, 'open' and an inactive, 'closed' conformation by the introduction of disulphide bonds and these domains were expressed as IgG Fc fusion proteins. αE fusion proteins recognize E-cadherin, the only known ligand for αEß7. This interaction was inhibited by an antibody that blocks the αE-binding site on E-cadherin and by the omission of Mn(2+) , which is essential for integrin function in vitro. The locked 'open' conformation of αE adhered to human oral and skin keratinocytes, including the E-cadherin-negative H376 cell line, and this was not inhibited by blocking antibody against the αEß7-binding site on E-cadherin, providing further evidence for the existence of an alternative ligand for αEß7 in skin and oral mucosa. The interaction with E-cadherin and the alternative ligand was Mn(2+) dependent and mediated by the metal ion-dependent coordination site (MIDAS) of the locked 'open'αE I domain, independently of the ß7 subunit.

MicroRNA-124 suppresses oral squamous cell carcinoma motility by targeting ITGB1.

Alterations in the levels of molecules which interact with the extracellular matrix, such as integrins, are associated with invasion of oral squamous cell carcinomas (OSCC). The molecular mechanisms underlying dysregulation of integrin expression in OSCC, however, remain unclear. Here, we show that microRNA-124, a small non-coding RNA down-regulated in OSCC, is able to downregulate expression of integrin beta-1 (ITGB1) by interacting with its 3' untranslated region. Over-expression of miR-124 attenuates endogenous ITGB1 expression and reduces the adherence and motility of OSCC cells, suggesting disruption of miR-124-mediated repression of ITGB1 may be a key factor in OSCC progression.

Functional expression of the chemokine receptor XCR1 on oral epithelial cells.

Chemokines are chemoattractant cytokines which act on specific receptors and play an important role in leukocyte migration as well as physiological and pathological processes. We investigated the role of the chemokine receptor XCR1 and its ligand lymphotactin (Lptn/XCL1) in the regulation of oral epithelial cell behaviour. In vitro XCR1 mRNA and cell surface protein expression was detected in normal oral keratinocytes and oral squamous cell carcinoma cell lines. Lymphotactin mediated intracellular activation of the ERK1/2 signalling pathway and stimulated migration, invasion, and proliferation of all cells through XCR1. Oral cancer cells showed a greater response to lymphotactin than normal keratinocytes and a direct relationship between receptor expression and migration, invasion, and proliferation was observed. Exposure of normal keratinocytes to lymphotactin resulted in increased adhesion to fibronectin but not collagen and stimulated MMP-2 and MMP-9 but not MMP-7 release, whereas exposure of cancer cells resulted in increased adhesion to both collagen and fibronectin and stimulated production of MMP-2, MMP-9, and MMP-7. We observed XCR1 but not lymphotactin to be expressed by epithelial cells in normal oral mucosa in vivo, whilst both were expressed and up-regulated in inflammatory oral disease and oral cancer including primary and metastatic disease. Lymphotactin mRNA and constitutive intracellular protein were detected in normal keratinocytes and oral cancer cell lines in vitro. These findings show that XCR1 and its ligand, lymphotactin, are expressed by oral epithelial cells and suggest that they play a role in regulating the behaviour of these cells.

Force generation and protease gene expression in organotypic co-cultures of fibroblasts and keratinocytes.

Fibroblast-epithelium interactions are crucial for successful tissue engineering of skin and oral mucosal equivalents. In this study, we assessed early force generation in organotypic fibroblast-epithelium co-cultures, using normal human keratinocytes (NHK) and HPV16-transformed (UP) cells. During the initial 2 h period, organotypic co-cultures containing both epithelial cell types produced significantly more force than fibroblasts alone (p < 0.05). After 2 h, the epithelial contribution became diminished and did not significantly contribute to intrinsic force generation by fibroblasts, and no differences were observed when using UP vs. NHK. We then measured protease gene expression at the end of the experimental period. Distinct differences were evident in protease expression both between NHK-human skin fibroblast (HSF) vs. UP-HSF co-cultures and compared to fibroblasts alone. We conclude that whilst the very early contractile response of fibroblasts is enhanced by the overlying epithelium, this becomes diminished as the fibroblast response becomes predominant and it does contribute to tissue remodelling via regulation of protease expression.

The 120 kDa cell-binding fragment of fibronectin up-regulates migration of alphavbeta6-expressing cells by increasing matrix metalloproteinase-2 and -9 secretion.

Changes in the extracellular matrix, integrin expression, and protease secretion occur in wound healing and cancer and these systems are thought to play a crucial role in such processes. In this study, experiments were performed to examine the interaction of epithelial cells with the 120 kDa cell-binding fibronectin fragment. Cell migration was significantly increased in response to the 120 kDa fragment when compared with the full-length molecule, but only in cells overexpressing the beta-6 integrin (VB6). This involved the up-regulation of matrix metalloproteinases (MMPs) 2 and 9, the levels of which are increased in the supernatant from VB6 cells plated on the 120 kDa fragment. Inhibition of MMP activity with both tissue inhibitor of metalloproteinase-1 (TIMP-1) and a chemical inhibitor (Ro32-3580) resulted in a significant reduction in migration on the fibronectin fragment. In vitro this fragment can be generated by incubating full-length fibronectin with purified MMP-9. Expression of the beta6 integrin subunit, which is only seen in wound healing and cancer, enhances the motility of and MMP secretion by epithelial cells in response to the 120 kDa fibronectin fragment. These enzymes can then further degrade fibronectin to generate additional fragments, leading to the creation of a positive feedback loop, which may be of significance in disease processes.

A recombinant fragment of the fibronectin-binding protein of Staphylococcus aureus inhibits keratinocyte migration.

Staphylococcus aureus is a common wound-infecting organism which can interact with cells via the extracellular matrix protein fibronectin (FN). The aim of this study was to determine the effect of the FN-binding protein (FnBP) of S. aureus on the behaviour of a human skin keratinocytes cell line (UP). FN-coated plates and Transwell membranes were exposed to a recombinant protein encompassing the D1-D4 repeat region of S. aureus FnBPB (rFnBPBD1-D4) before cell adhesion and migration assays. The influence of this protein on migration into a wounded area, cell cycle progression and endogenous cellular FN assembly was also assessed. The rFnBPBD1-D4 protein potently inhibited S. aureus entry into UP keratinocytes but had no effect on cell adhesion to FN substrate. It inhibited UP keratinocyte Transwell migration by 17% (P= 0.04) and 31% (P=0.02) at 10 and 100 microg/ml, respectively. In the wound assay, 100 microg/ml of rFnBPBD1-D4 protein reduced the migration area by approximately 30%. No differences in cell cycle progression were observed. In the presence of rFnBPBD1-D4, most of the cellular FN matrix on the cell surface and along the cell filopodia seen in untreated cells was absent. Interaction of S. aureus FnBPs with FN may influence cell behaviour and thus play a role in delayed epithelial closure in infected healing wounds.

Internalization of Staphylococcus aureus by human keratinocytes.

Staphylococcus aureus is among the most important human pathogens and causes various superficial and systemic infections. The ability of S. aureus to be internalized by, and survive within, host cells, such as keratinocytes, may contribute to the development of persistent or chronic infections and may finally lead to deeper tissue infections or dissemination. To examine the mechanisms of internalization of S. aureus by keratinocytes, isogenic mutants lacking fibronectin-binding proteins (FnBPs), a recombinant protein consisting of the fibronectin-binding domain of S. aureus FnBPs, and an anti-alpha5beta1 antibody were used in cocultures with immortalized keratinocytes and primary keratinocytes. We found that internalization of S. aureus by immortalized keratinocytes requires bacterial FnBPs and is mediated by the major fibronectin-binding integrin alpha5beta1. In contrast to internalization by immortalized keratinocytes, internalization of S. aureus by primary keratinocytes could occur through FnBP-dependent and -independent pathways. S. aureus clumping factor B (ClfB), which was recently determined to bind to epithelial cells, was not involved in the uptake of this bacterium by keratinocytes. The identification of an alternate uptake pathway, which is independent of S. aureus FnBPs and host cell alpha5beta1, has important implications for the design of therapies targeted to bacterial uptake by host cells.

Modulation of the urokinase-type plasminogen activator receptor by the beta6 integrin subunit.

Over-expression of components of the urokinase system is well documented in cancer and is thought to enable tumour cells to migrate and invade. Changes in integrin expression are also a common feature of tumours and have been linked to changes in protease activity. It has been shown that the alphavbeta6 integrin is neo-expressed in a number of epithelial carcinomas and in wound healing situations. We therefore investigated whether alphavbeta6 is able to modulate a key regulator of proteolysis, the urokinase receptor. We report that epithelial cells expressing full-length alphavbeta6 exhibit decreased urokinase receptor expression and function. Furthermore, this novel modulation requires the C-terminal 11 amino acids of the cytoplasmic tail of the beta6 integrin subunit. Cells expressing alphavbeta3, however, did not affect urokinase receptor expression. De novo expression of beta6 by melanoma cells and beta3 by epithelial cells did not influence urokinase receptor expression or function, suggesting that modulation of urokinase system is both integrin subunit and cell-specific.

Functional regulation of tissue plasminogen activator on the surface of vascular smooth muscle cells by the type-II transmembrane protein p63 (CKAP4).

We have demonstrated that tissue plasminogen activator (tPA) binds specifically to human vascular smooth muscle cells (VSMC) in a functionally relevant manner, both increasing plasminogen activation and decreasing tPA inhibition (Ellis, V., and Whawell, S. A. (1997) Blood 90, 2312-2322; Werner, F., Razzaq, T. M., and Ellis, V. (1999) J. Biol. Chem. 274, 21555-21561). To further understand this system we have now identified and characterized the protein responsible for this binding. Rat VSMC were surface-labeled with 125I, and cell lysates were subjected to an affinity chromatography scheme based on the previously identified tPA binding characteristics. A single radiolabeled protein of 63 kDa bound specifically and was eluted at low pH. This protein was isolated from large scale preparations of VSMC and unambiguously identified as the rat homologue of the human type-II transmembrane protein p63 (CKAP4) by matrix-assisted laser desorption ionization and nano-electrospray tandem mass spectrometry of tryptic fragments. In confirmation of this, a monoclonal antibody raised against authentic human p63 recognized the isolated protein in Western blotting. Immunofluorescence microscopy demonstrated that p63 was located principally in the endoplasmic reticulum but was also detected in significant quantities on the surface of human VSMC. In support of the hypothesis that p63 is the functional tPA binding site on VSMC, an anti-p63 monoclonal antibody was found to block tPA binding. Furthermore, heterologous expression of an N-terminally truncated mutant of p63, which targets exclusively to the plasma membrane, led to an increase in tPA-catalyzed plasminogen activation. Therefore, p63 on the surface of VSMC may contribute to the functional regulation of the plasminogen activation system in the vessel wall.